This invention relates to allergenic proteins of natural rubber latex in substantially purified form, their production and their use, together with monoclonal antibodies developed against those allergenic proteins, in assays for the qualitative and quantitative determination of the levels of the allergenic proteins in natural rubber latex or in products made from latex. Assays for identifying and/or quantitating antibodies in blood or blood products that mediate the occurrence of an allergic reaction induced by natural rubber latex are also provided, together with in vivo and in vitro diagnostic tests for detecting hypersensitivity to natural rubber latex and which involve use of the aforesaid allergenic proteins. The invention also provides for the use of the aforesaid allergens as de-sensitising agents in the treatment of latex protein allergy. There is still further provided a method for removing allergenic proteins from latex products.
There now follows a glossary defining certain of the terms used hereinafter:
Total protein: All protein and fragments thereof present in a sample.
Antigenic proteins: A group within total proteins. These proteins elicit antibody production in the animal and human body. The antibodies elicited might include those of the IgE class which are able to induce an allergic reaction and also those that do not induce allergy. xe2x80x9cAntigenic proteinsxe2x80x9d can also refer to proteins that are recognised by (react with) antibodies.
Allergenic proteins: A group within antigenic proteins (and hence a sub-group within total proteins). These proteins elicit production of the IgE class of antibodies in the animal or human body. They can induce an allergic reaction where IgE specific to them are present. xe2x80x9cAllergenic proteinsxe2x80x9d can also refer to proteins that are recognised by (react with) IgE.
Allergens: Substances (proteins or otherwise) that elicit production of the IgE class of antibodies in the animal or human body. They can induce an allergic reaction where IgE specific to them are present. xe2x80x9cAllergensxe2x80x9d can also refer to substances that are recognised by (react with) IgE.
With regard to latex protein allergy, the only known allergens are proteinaceous in nature. Hence, in this context, the terms latex allergens, allergens, allergenic proteins and protein allergens are synonymous.
Antibodies: Immunoglobulins present in the serum of an animal and synthesised by plasma cells in response to an antigen.
IgE: A group within antibodies. IgE specific for an allergen is elicited in the animal or human body by its exposure to the allergen. Subsequent exposure to the allergen may induce an allergic reaction.
Polyclonal antibodies: A collection of antibodies against a particular antigen. Since most antigens have a large number of epitopes, there can be many different antibodies against a given antigen.
Monoclonal antibody: An immunoglobulin (antibody) produced by a single clone of lymphocytes. A monoclonal antibody recognizes only a single epitope on an antigen.
Epitope: An antigenic determinant in a molecule which is specifically recognised by an antibody combining site or by the antigen receptor of a T-cell.
Hybridoma: A cell line obtained by the fusion of a myeloma cell line, which is able to grow indefinitely in culture, with a normal antibody secreting B-cell. The resulting cell line has the properties of both partners, and continues to secrete the antibody product of the normal B-cell. By selecting a myeloma that has ceased to make its own immunoglobulin product, but has retained the machinery for doing so, the hybridoma secretes only the normal B-cell antibody. Since the cell line is cloned, the antibody is monoclonal.
Allergenic proteins (allergens) can induce an allergic reaction in sensitised persons which, in severe cases, can lead to an anaphylactic shock that is potentially lethal. Proteins present in latex products, such as latex gloves, can induce a form of allergy known as xe2x80x9cType I hypersensitivityxe2x80x9d in a small proportion of people who use such products. The use of rubber products, especially dipped latex products, is therefore being viewed with some caution and concern from the healthcare viewpoint.
Natural rubber from the commercial rubber tree, Hevea brasiliensis, is an important commodity in the economies of many Asian and African countries. Natural rubber is marketed in the form of bales, sheets and as latex concentrate. A major demand for natural rubber latex concentrate is in the manufacture of xe2x80x9cdipped-latexxe2x80x9d products such as gloves for examination, surgical and domestic use. In 1993, Malaysia alone exported latex dipped goods valued at a total of US$880 million. The global demand for latex examination gloves in particular has increased significantly in recent years with the rise in the incidence of the Acquired Immuno-deficiency Syndrome (AIDS) due to HIV infection.
There have been recent reports that gloves and other surgical aids manufactured from natural rubber latex can cause contact urticaria which, in a few cases, has led to anaphylactic reactions in persons previously sensitised (Nutter, 1979; Turjanmaa et al., 1984; Axelsson et al., 1987; Leynadier et al., 1989). Anaphylaxis can be life-threatening and is therefore far more serious than the generally mild skin sensitivities caused by the various chemical compounds used in glove manufacture. While contact dermatitis arising from chemicals has been recognized for many years, the allergic response to proteins in latex products such as gloves and catheters poses a potentially serious threat to their users. Those most at risk are health care workers, who may wear latex gloves more or less continuously throughout their working day, and their patients. Ultimately, the product manufacturers and the latex industry as a whole will feel the repercussions from the perceived threat, even though the proportion of people that are actually at risk is very small. Regulatory agencies such as the US Food and Drug Administration (FDA) have already indicated that they will require all natural latex goods to be identified as such in the near future. The US FDA may also soon be setting standards for acceptable levels of total protein in latex products. If this problem is not addressed with the urgency it deserves and means established to distinguish between xe2x80x9csafexe2x80x9d and xe2x80x9cunsafexe2x80x9d products, it is even possible that future legislation could impose a blanket ban on the use of all latex products in medical care.
Latex protein allergy has therefore been regarded with increasing concern in recent years, especially by the manufacturers of latex products and by those involved in healthcare. Evidence has pointed to water-extractable proteins in latex as the cause of the latex-induced anaphylactic reaction which is mediated through an interaction between allergenic latex proteins and a class of antibodies (IgE), in the sensitised person. IgE specific immunoassays of protein fractions have suggested that more than one specific protein may be involved (Turjanmaa et al. 1988; Slater, 1991).
In view of the importance of the latex protein allergy problem, both from the healthcare perspective and from the viewpoint of latex product manufacturers, active research in this connection is being undertaken in various laboratories worldwide. The main objectives of the investigations are:
(a) To produce a latex concentrate containing lower levels of allergens. Latex concentrate suppliers are seeking to reduce allergens in the source material used for the manufacture of latex products.
(b) To manufacture a low allergen latex product. Latex product manufacturers are seeking to reduce allergens in their finished products.
(c) To develop an assay for the quantitation of allergens present in latex concentrate or in products. Both the latex suppliers as well as latex product manufacturers require an assay for the purpose of standardisation and quality control to complement (a) and (b).
In the conventional preparation of latex concentrate, field latex is stabilised with ammonia (to prevent flocculation or coagulation of the rubber) and then concentrated by centrifugation to increase the rubber content from about 33% to 60%. Currently, there are two main approaches to the production of a low protein latex concentrate (Subramaniam, 1992). Firstly, multiple centrifugations can be carried out with fresh ammoniated water added in each cycle to dilute out the soluble proteins in the aqueous phase of the latex. Secondly, the latex can be treated with a protein-degrading enzyme (proteinase).
To manufacture latex-dipped products with reduced soluble proteins, the simplest method is to wash in water. Proteins migrate to the surface of the latex film as it dries (Shamsul Bahri et al., 1993) and are hence most effectively removed when the film is washed after complete drying.
While the above-mentioned measures are aimed at reducing the amount of allergens in the latex concentrate or in the finished latex product, there is at present no reliable way to assess the level of such allergens. In the absence of a specific latex allergen assay, samples are currently tested for total protein on the assumption that low total protein levels would be an indication of low allergen levels. This is generally true when comparing extreme protein levels (i.e. very high protein levels are associated with high allergenicity and very low protein levels are associated with low allergenicity). However, total protein levels can sometimes give a misleading picture as to how potentially harmful a product can be because not all proteins are allergenic (i.e. able to induce an allergy); many are innocuous. A better assay would be one that estimates the level of the latex allergens specifically.
Assays for latex allergens are commercially available and come in a number of variations, but they are all based on one or more immunological reactions of an immunoassay. An immunoassay is based on the interaction between antibodies with the specific protein antigens to which the antibodies will bind. Hence, in the case of an immunoassay for latex allergens, the principal reaction is the binding of latex allergens with the antibodies that recognise such allergens.
Assays based on the immune reaction relating to the latex allergy problem fall into two categories. In the first category, the assays quantitate the antibodies (IgE) in a blood sample that mediate the occurrence of an allergic reaction. Essentially, such assays are for healthcare and medical use and they serve as diagnostic tests for latex allergy. The second category of immunoassays pertains to the quantitation of latex allergens extractable from latex products. Essentially, such assays are for use in the latex industry to monitor and regulate the levels of latex allergens in latex concentrate and in manufactured latex products. Presently, there are a small number of commercially available immunoassays for both categories of tests.
Commercial immunoassays in RAST (radioallergosorbent test) and ELISA (Enzyme-linked immunosorbent assay) formats are used to determine if specific IgE to the latex proteins is present and, hence, test for latex allergy in patients. Such tests can also be performed as competitive assays (RAST inhibition, competitive ELISA) to quantitate the amount of allergen in a latex sample or a latex product.
All these immunoassays require the use of specific latex allergens in the immunological reaction. Since no latex allergens have so far been isolated and conclusively identified, the presently available commercial assays use as their allergen source crude (unpurified) latex serum or proteins eluted from commercially manufactured latex gloves.
Crude latex serum is unreliable as a source of allergens, however, because it contains a lot of proteins (and other substances) other than the allergens themselves. These impurities might interfere with the precision of the assay. There is also no information as to the level or consistency of the allergens in latex obtained from different sources, from different times of the year or latex that has been preserved or stored under different conditions. While the variation in allergenic protein levels in different batches of latex has not been investigated, latex proteins in general, and latex enzymes (a class of proteins) in particular, are known to vary with clonal (cultivar) source, season, physiological state of the tree, the intensitiy of tapping (latex harvesting) and the use of chemical stimulants to promote latex yield. For example, differences in latex proteins sourced from different commercial clones were discerned after electrophoretic separation (Walujono and Suseno 1973, Yeang et al., 1977, Prematillake and Yapa, 1985), indicating that protein composition varied between clones. The fact that some of these differences could be traced to latex B-serum proteins (Yeang et al., 1977) is significant in view of the fact that, as has now been discovered by the inventors of the present invention, some of the major latex allergens originate from the B-serum. Latex protein composition could also be influenced by the physiological state of the tree. Prematillaka et al. (1985) reported the disappearance or reduction of a number of latex proteins collected from trees afflicted with the physiological disorder known as brown bast.
Differences in the iso-forms of enzymes of latex obtained from various commercial Hevea clones have also been demonstrated (Chevallier, 1988). The activities of certain latex enzymes vary significantly with season (Yeang and Paranjothy, 1982). Moreover, latex enzyme activities are known to change significantly in response to the intensity of latex harvest (Yeang and Paranjothy, 1982a) and to yield stimulation by the chemical ethephon (Tupy, 1969; Chrestin et al., 1985). The level of a latex protein complex known as a microhelix has been reported to increase in the B-serum as a result of ethephon stimulation (Gomez and Moir, 1979). The microhelix has also been shown to be very variable and sometimes undetected in B-serum. This variation occurs between clones and also between samples taken at different times from the same group of trees (Gomez and Moir, 1979; Gomez and Tata, 1977). The last mentioned point is significant in view of the finding described below that one of the identified latex allergens is a component of the microhelix complex.
As mentioned above, proteins eluted from commercial latex gloves are also used as the protein antigen component of immunoassays for the diagnosis of latex allergy or for the quantitation of latex allergens. A serious drawback of this approach is that different brands of gloves (or even different batches of the same brand) show qualitative and quantitative differences in their allergen composition. As such, test results from assays that utilise latex glove proteins as antigens can vary considerably depending on the choice of latex gloves from which the antigens were sourced.
Not surprisingly, the commercially available latex allergen assays lack sensitivity and specificity and are only partially successful in detecting allergenicity.
Working with a commercially available latex antigen preparation, (supplied by Stallergenes), Levy (1993) reported that it gave positive results in 100% of sensitive patients and negative results in non-sensitive control patients. In another study (Lagier et al., 1992), however, 80% of test patients (nurses) who were known to be allergic to latex gave negative results with the commercial Stallergenes kit. From a study of forty allergic patients (diagnosed by skin prick tests), Leynadier, Autegard and Levy (1993) reported 5-16% false negative results with Stallergenes latex allergen as well as two other commercial allergens, supplied by Allerbio and Bencard. Hence, false negative results occur with the commercial latex allergens currently available. The most widely used RAST kit is probably the latex RAST k82 produced by Pharmacia Diagnostics and its enzyme-linked immunosorbant assay (Pharmacia CAP system). Levy (1993) reported that these assays were capable of detecting IgE antibody in the serum of 40-90% of skin prick test-positive latex-allergic patients.
Another commercially available immunoassay for latex allergens is the xe2x80x9cLatex ELISA for Antigenic Proteinsxe2x80x9d (LEAP) manufactured by the Guthrie Research Institute, U.S.A. (Beezehold, 1993). The assay is based on an indirect ELISA (enzyme-linked immunosorbent assay) in which polyclonal antibodies against latex proteins are used. Such an assay might not be sufficiently effective in discriminating latex protein antigens in general (i.e. proteins that bind both to the allergy-inducing antibodies (IgE) and the non allergy-inducing antibodies) from the latex allergens (i.e. proteins that bind to IgE specifically). The use of this assay also assumes that all antigens and allergens bind equally well to the ELISA plate under the same conditions since the test samples containing the antigens/allergens to be determined are required to be bound to the ELISA plate by the end-user using a single set of conditions. This assumption may be false and therefore antigens and allergens not able to bind well to the plate under the conditions used will not be detectable or at best sub-optimally detectable.
There is therefore a need for an improved latex allergen assay. In order to produce such an assay antibodies against the individual latex allergens must be developed and be made available. In order to do that, the specific latex allergens must first be identified.
There have been many publications reporting on the occurrence of various latex allergenic proteins. Practically all the references to latex allergens characterise the proteins by molecular weight and/or occasionally by their isoelectric point. Latex allergens characterised by molecular weight or isoelectric point alone cannot be regarded as having been identified because:
(a) Proteins break down during the manufacture of latex products and a single allergenic protein can hence appear as several proteins of lower molecular weights in a protein separation procedure such as gel filtration, HPLC, isoelectric focussing or electrophoresis to determine molecular weight or isoelectric point.
(b) Proteins may aggregate to form protein complexes that have different apparent molecular weights and isoelectric points from those of unaggregated proteins.
and
(c) Several different proteins may have similar characteristics (e.g. molecular weight) and cannot therefore be easily distinguished from one another. According to the findings of the inventors of the present invention, these difficulties are overcome by:
(i) Isolating specific latex allergenic proteins from natural rubber latex.
(ii) Developing monoclonal antibodies against those specific allergenic proteins to tag them and identify their break-down protein fragments and sub-units.
According to the present invention, three specific latex allergens have been identified. The allergens have been designated Hev b IV, Hev b II and Hev b III in accordance with the allergen nomenclature system approved by the International Union of Immunological Societies and published in the Bulletin of the World Health Organisation (Marsh et al., 1986; Marsh, 1987). The protein Hev b IV was originally designated Hev b I by the present inventors, but has been re-designated Hev b IV because Hev b I has previously been assigned by other workers to the latex protein known as Rubber Elongation Factor found on the surface of rubber particles. (Czuppon et al).
Monoclonal antibodies have been generated against all three allergens and, significantly, some of these monoclonal antibodies also recognise the breakdown products or sub-units of the allergens. Assays are also provided according to the present invention and which are based on the interaction between the aforesaid specific allergenic proteins isolated from natural rubber latex and monoclonal antibodies developed against those proteins.
More specifically, the present invention provides an allergenic protein of natural rubber latex (designated Hev b IV) characterised by being in a substantially purified form and which is oligomeric and consists of three major species of monomeric polypeptides with molecular weights 50, 55 and 57 kDa that are disulphide-linked into dimers of approximate molecular weights 100, 110 and 115 kDa, and allergenic sub-units or aggregates thereof.
The invention further provides a second allergenic protein of natural rubber latex (designated Hev b II) characterised by being in a substantially purified form and which is composed of two polypeptide claims of molecular weights 34/35 kDa and 36/37 kDa, and allergenic sub-units or aggregates thereof.
The invention still further provides a third allergenic protein of natural rubber latex (designated Hev b III) characterised by being in a substantially purified form and which has a molecular weight of 24 kDa, and allergenic sub-units or aggregates thereof.
In another aspect, the invention provides monoclonal antibodies developed against the aforesaid allergens Hev b IV, Hev b II and Hev b III.
The invention further provides a method for the production of the aforesaid allergenic proteins Hev b IV, Hev b II and Hev b III.
In a still further aspect the invention provides assays for the qualitative and quantitative determination of the levels of allergens of natural rubber latex which are based on the interactions between specific protein allergens isolated from natural rubber latex or other tissue of the rubber tree Hevea brasiliensis and monoclonal antibodies developed against those allergens. The allergens being assayed may be present in the latex intended for use in the manufacture of latex products or they may be present in manufactured latex products. The assays can also be used to quantitate allergenic latex proteins in products made from dry rubber.
The invention further provides for the application of some or all of the same antibody-allergen interactions to identify and/or quantitate antibodies that mediate the occurrence of an allergic reaction induced by natural rubber latex. Such antibodies belonging to the class of antibodies known as IgE are normally found in blood or blood products.
In yet another embodiment of the invention, latex allergenic proteins and/or monoclonal antibodies against such proteins and/or a mixture thereof are labelled, for example with biotin, in order that their presence can be detected when they are used in an assay.
The invention provides also for the use of the aforesaid allergens Hev b IV, Hev b II and Hev b III, in in vivo or in vitro (ex vivo) diagnostic tests for the determination of Type I hypersensitivity to natural rubber latex (for example, the skin prick test or the histamine release test).
The invention also provides for the use of the aforesaid allergens, Hev b IV, Hev b II and Hev b III, as de-sensitising agents in the treatment of latex protein allergy.
Another embodiment of the invention provides for the leaching or washing of a latex product in a solution of salt (e.g. sodium chloride) or other solution with ionic strength greater than that of water to selectively remove latex allergens known to be soluble in solutions of ionic strength higher than that of water.
The inventors believe that they are the first to establish clearly the identities and properties of the three latex proteins, the first to report a reproducible method for their purification in large quantities from fresh latex and the first to demonstrate the allergenic nature of these proteins and to propose their use in assays. They have also developed monoclonal antibodies against these proteins. It is believed that Hev b II was completely unknown before the present invention. While the existence of Hev b III and Hev b IV had already been suggested, they were identified only to the level of their molecular weight or isoelectric point. Theoretically, proteins can be extracted from latex on the basis of their molecular weight even without knowing which component of latex the proteins originate from and without any other information. In practice, however, it is not easy to extract and purify specific proteins based on molecular weight alone with any reasonable assurance of the absence of contamination by proteins with similar characteristics (i.e. molecular weight) that co-purify with the desired allergenic proteins. As will be appreciated, this would make it difficult to aliquot out the precise quantities of pure allergens that would be required for quantitative immunological assays.
Results from experiments carried out suggest that the three specific allergenic proteins identified by the present inventors, designated Hev b IV, Hev b II and Hev b III, and their breakdown products or sub-units, account for a very large proportion of the latex protein allergens that have previously been reported.
Monoclonal antibodies are very specific for the particular allergens and antigenic sites (epitopes) that they recognise. Using an appropriate monoclonal antibody that recognises an epitopic site that is present in the breakdown fragments and sub-units of the parental allergen, the identities of such fragments and sub-units can be elucidated.
Besides their importance in identifying protein allergens and their breakdown fragments and sub-units, the monoclonal antibodies are also important for the purpose of developing commercial allergen assays in that (as compared with polyclonal antibodies) they lend themselves to sustained production in a consistent form and on the scale required for commercialisation.
Yet a further advantage of monoclonal antibodies is that they can be incorporated into a system of protein purification known as xe2x80x9caffinity chromatographyxe2x80x9d. Using such a system, the allergens that the monoclonal antibodies recognise can be isolated and purified in relatively large quantities, sufficient for their use in one variation of the allergen assay, known as a xe2x80x9ccompetitive binding assayxe2x80x9d, in which both the antibodies and antigens are required. Pure allergens may also be used in immunological assays to quantitate the latex-induced antibodies (IgE) in a blood sample, or for use in skin prick tests for diagnostic purposes.
According to the present invention, selected monoclonal antibodies developed against the allergens Hev b IV, Hev b II and/or Hev b III are used in the development of immunoassays for the quantitation of allergens or the quantitation of latex specific IgE. Three monoclonal antibodies that recognise the latex allergens Hev b II, Hev b III and Hev b IV are USM/RB4, USM/RC2 and USM/RB3 respectively.
Immunoassays that incorporate the use of specific allergens are superior to immunoassays where the allergens are contained in unfractionated latex serum. In the latter, the exact allergen level in the serum is unknown while the consistency of the allergen in latex sera from different sources is also uncertain.
Further, immunoassays that incorporate the use of monoclonal antibodies specific for allergens are superior to those using polyclonal antibodies in immunoassays where whole latex sera, allergen-enriched or semi-purified antigen preparations are used instead of purified specific allergens.
Examples of immunoassays which form part of this invention are:
(a) A competitive binding assay in which the test sample is used to inhibit binding of labelled specific allergens to a solid phase bearing either a monoclonal antibody or antibodies, or a polyclonal antiserum or antibodies.
(b) A two-site assay in which polyclonal antibodies bound to a solid phase are used to xe2x80x9ccapturexe2x80x9d specific allergen(s) and the presence of the captured allergen detected by a monoclonal antibody which may or may not be labelled directly.
(c) A two site-assay in which monoclonal antibodies bound to a solid phase are used to xe2x80x9ccapturexe2x80x9d specific allergen(s) and the presence of the captured allergen detected by a polyclonal antibody which may or may not be labelled directly.
(d) A two-site assay in which monoclonal antibodies bound to a solid phase are used to xe2x80x9ccapturexe2x80x9d specific allergen(s) and the-presence of the captured allergen detected by other monoclonal antibodies which may or may not be labelled directly.
(e) Any form of immunoassay in which the specific allergens Hev b IV, Hev b II and Hev b III are used bound or in solution as in nephelometry, radioallergosorbant assays (RAST) or RAST inhibition assays.